Over the years, numerous synthetic approaches have been utilized for covalently attaching a water soluble polymer such as a polyethylene glycol to a biologically active agent. Early attempts at PEGylation typically resulted in non-specific covalent attachment of PEG to multiple reactive sites in the active agent, most commonly a polypeptide or protein. In the case of polypeptides, the most common reactive groups for covalent coupling are the alpha or epsilon amino groups of lysine. Early PEGylation chemistry typically employed low molecular weight, linear PEG reagents, and most conjugates were produced via acylation (Roberts, M. J., et al., Advanced Drug Delivery Reviews, 54 (2002), 459-476). Such early PEGylated drugs often resulted in poor drug performance, and/or exhibited low batch to batch reproducibility.
Second generation PEGylation chemistry resulted in approaches designed to overcome many of the problems associated with first generation PEGylation reagents—diol contamination, side reactions, unstable linkages, and lack of selectivity in covalent attachment of the polymer to multiple reactive sites in the active agent (Roberts, M. J., et al., ibid). New PEG reagents such as PEG propionaldehyde (U.S. Pat. No. 5,252,714), PEG vinyl sulfone (U.S. Pat. No. 5,446,090) and PEG maleimide (U.S. Pat. No. 6,602,498), among others, were developed and methods involving their use described. Non-linear PEGs such as branched (U.S. Pat. No. 5,932,462) and forked PEGs (U.S. Pat. No. 6,437,025) also became more widely available, e.g., from companies such as Nektar Therapeutics and NOF.
As PEGylation of therapeutics has become more widely employed as one possible approach for improving the pharmacological and other properties of therapeutic agents, the synthetic challenges for consistently forming a well-defined PEGylated therapeutic having beneficial pharmacological properties continue to mount. Such approaches, particular if they are to be adapted to large scale production, should ideally avoid multiple reaction steps, as well as minimize the number of protection, deprotection, and purification steps required, as well as form product in a consistent manner and with reasonably good yields.